Fatty alcohols and fatty acid esters useful for treatment of inflammation

ABSTRACT

Immunomodulators selected from: (a) a saturated or cis-unsaturated C 10 -C 20  fatty alcohol or an ester thereof with a C 1 -C 6  alkanoic acid; (b) a monoester of a C 2 -C 8  alkanediol or of Glycerol with a saturated or cis-unsaturated C 10 -C 20  fatty acid; and (c) a diester of glycerol with a saturated or cis-unsaturated C 10 -C 20  fatty acid, are useful for treatment of inflammation, particularly immunologically-mediated inflammation such as it occurs in autoimmune diseases.

FIELD OF THE INVENTION

The present invention relates to anti-inflammatory agents and, moreparticularly, to fatty alcohols, esters thereof with C₁-C₆ alkanoicacids or esters of fatty acids with alkanediols or glycerol which areuseful in the treatment of immunologically-mediated inflammation.

Abbreviations: AA: adjuvant arthritis: CFA: complete Freund's adjuvant;EAE: experimental autoimmune encephalomyelitis; GPSCH: guinea pig spinalcord homogenate; IFA: incomplete Freund's adjuvant; OA: oleyl alcohol;PBS: phosphate-buffered saline; SC: subcutaneously.

BACKGROUND OF THE INVENTION

Inflammation is commonly divided into three phases: acute inflammation,the immune response and chronic inflammation. Acute inflammation is theinitial response to tissue injury and is mediated by the release ofhistamine, serotonin, bradykinin, prostaglandins and leukotrienes. Theimmune response, usually preceded by the acute inflammation phase,occurs when immunologically competent cells are activated in response toforeign organisms or antigenic substances liberated during the acute orchronic inflammatory response. The outcome of the immune response forthe host may be beneficial, as when it causes invading organisms to bephagocytosed or neutralized. However, the outcome may be deleterious ifit leads to chronic inflammation without resolution of the underlyinginjurious process as it occurs in rheumatoid arthritis.

The treatment of patients with inflammation envisages the relief ofpain, which is the presenting symptom and the major continuing complaintof the patient, as well as the slowing or arrest of the tissue-damagingprocess.

Anti-inflammatory agents are usually classified as steroidal orglucocorticoids and nonsteroidal anti-inflammatory agents (NSAIDs). Theglucocorticoids are powerful anti-inflammatory agents but the hightoxicity associated with chronic corticosteroid therapy inhibits theiruse except in certain acute inflammatory conditions. Therefore, thenonsteroidal anti-inflammatory drugs have assumed a major role in thetreatment of chronic conditions such as rheumatoid arthritis.

Among the nonsteroidal anti-inflammatory agents are included derivativesof aminoarylcarboxylic acids, arylacetic acids, arylbutyric acids,arylcarboxylic acids, arylpropionic acids, pyrazole, pyrazolone,salicylic acid and some other derivatives of different chemicalstructure, including specific anti-arthritic/anti-rheumatic agents.

Some fatty alcohols and esters of fatty acids have been described assolvents or emulsifiers for use in pharmaceutical compositions. Forexample, cetyl alcohol may be used in pharmaceutical compositions asemulsifying and stiffening agent (The Merck Index, pp. 347-8, # 2037),oleyl alcohol may be used as a carrier for medicaments (The Merck Index,p. 1222, # 6900), and alkyl esters of oleic acid may be used as solventsfor medicaments (The Merck Index, p. 6899, # 6898).

A mixture of higher aliphatic primary alcohols, primarily isolated frombeeswax, was described as having moderate anti-inflammatory activity.The composition of such a mixture was not disclosed (Rodriguez et al.,1998).

Feeding laboratory animals with fish oil rich in the long-chain n-3polyunsaturated fatty acids (PUFAs), eicosapentaenoic acid (20:5n-3) anddocosahexaenoic acid (22:6n-3), was described to reduce acute andchronic inflammatory responses, to improve survival to endotoxin and inmodels of autoimmunity and to prolong the survival of grafted organs,and it was therefore suggested that fish oil supplementation may beclinically useful in acute and chronic inflammatory conditions andfollowing transplantation (Calder, 1998). A pharmaceutical preparationcomprising eicosapentaenoic acid and/or stearidonic acid for treatmentof schizophrenia is described in WO 98/16216 and U.S. Pat. No.6,331,568.

Modified polyunsaturated fatty acids and derivatives thereof have beenproposed for pharmaceutical uses. WO 99/27924 and U.S. Pat. No.6,280,755 describe anti-inflammatory fatty acids uninterrupted by amethylene group for use in topical pharmaceutical and cosmeticcompositions. WO 97/38688 and U.S. Pat. No. 6,262,119 describepolyunsaturated fatty acids having 1 or 2 substitutions selected fromoxa and thia in position beta or gamma to the acyl group, for treatingor ameliorating symptoms of T-cell mediated disease. WO 99/58122 andU.S. Pat. No. 6,365,628 describe saturated fatty acids in which one ormore methylene groups are substituted by O, S, SO, SO₂, or Se and alkylesters thereof, for treatment or prevention of diabetes. U.S. Pat. No.5,019,383 describes synthetic vaccines comprising a peptide residuecoupled to one or more alkyl or alkenyl groups of at least 12 carbonatoms or other lipophilic substance, wherein said alkyl or alkenyl groupmay be a fatty acid residue coupled to one or more functional groups ofa polyfunctional group which is bound to the N-terminal amino groupand/or C-terminal carboxy group of the peptide residue.

There is no description in the literature that isolated fatty alcoholsor esters thereof with alkanoic acids may be used themselves asmedicaments, and specifically not that they may be involved inimmunomodulation of inflammation.

SUMMARY OF THE INVENTION

It has now been surprisingly found, in accordance with the presentinvention, that certain long-chain fatty alcohols, esters thereof withC₁-C₆ alkanoic acids, or certain esters of long-chain fatty acids withalkanediols or glycerol can suppress inflammation in experimentaladjuvant arthritis (AA) and experimental autoimmune encephalomyelitis(EAE) models in rats and can prevent graft rejection in mice.

The present invention thus relates to pharmaceutical compositions forthe treatment of inflammation, particularly immunologically-mediatedinflammation, comprising as active ingredient an immunomodulatorselected from: (a) a saturated or cis-unsaturated C₁₀-C₂₀ fatty alcoholor an ester thereof with a C₁-C₆ alkanoic acid; (b) a monoester of aC₂-C₈ alkanediol or of glycerol with a saturated or cis-unsaturatedC₁₀-C₂₀ fatty acid; and (c) a diester of glycerol with a saturated orcis-unsaturated C₁₀-C₂₀ fatty acid.

In another embodiment, the invention relates to the use of animmunomodulator selected from: (a) a saturated or cis-unsaturatedC₁₀-C₂₀ fatty alcohol or an ester thereof with a C₁-C₆ alkanoic acid;(b) a monoester of a C₂-C₈ alkanediol or of glycerol with a saturated orcis-unsaturated C₁₀-C₂₀ fatty acid; and (c) a diester of glycerol with asaturated or cis-unsaturated C₁₀-C₂₀ fatty acid, for the preparation ofa pharmaceutical composition for the treatment of inflammation, inparticular immunologically-mediated inflammation.

In still another embodiment, the invention relates to a method for thetreatment of inflammatory disorders, in particularimmunologically-mediated inflammation, which comprises administering toan individual in need thereof an effective amount of an agent selectedfrom an immunomodulator selected from: (a) a saturated orcis-unsaturated C₁₀-C₂₀ fatty alcohol or an ester thereof with a C₁-C₆alkanoic acid; (b) a monoester of a C₂-C₈ alkanediol or of glycerol witha saturated or cis-unsaturated C₁₀-C₂₀ fatty acid; and (c) a diester ofglycerol with a saturated or cis-unsaturated C₁₀-C₂₀ fatty acid.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the dose response effect of oleyl alcohol (OA) on adjuvantarthritis (AA). Different doses of OA were administered subcutaneouslyto rats once 14 days before induction of AA.

FIG. 2 is a graph showing the disease profile of Lewis rats withexperimental autoimmune encephalomyelitis (EAE) and treated with oleylalcohol. Oleyl alcohol was administered to the rats 14 days beforeinduction of EAE. Control group was treated with incomplete Freund'sadjuvant (IFA).

FIG. 3 is a graph showing the disease profile of Lewis rats with EAE andtreated with IFA. IFA was administered to the rats 14 days beforeinduction of EAE. Control group was not treated.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides immunomodulators selected from: (a) asaturated or cis-unsaturated C₁₀-C₂₀ fatty alcohol or an ester thereofwith a C₁-C₆ alkanoic acid; (b) a monoester of a C₂-C₈ alkanediol or ofglycerol with a saturated or cis-unsaturated C₁₀-C₂₀ fatty acid; and (c)a diester of glycerol with a saturated or cis-unsaturated C₁₀-C₂₀ fattyacid.

According to one preferred embodiment of the invention, thepharmaceutical composition comprises a long-chain saturated orunsaturated C₁₀-C₂₀, preferably C₁₆-C₂₀, most preferably a C₁₈, fattyalcohol.

Examples of C₁₀-C₂₀ saturated fatty alcohols that can be used accordingto the invention include, but are not limited to, decyl alcohol, laurylalcohol, myristyl alcohol, stearyl alcohol and preferably cetyl alcohol(also known as palmityl alcohol).

The unsaturated fatty alcohol according to the invention has preferablyone or more double bonds in the cis form and 16-18 carbon atoms and maybe, without being limited to, oleyl alcohol (cis-9-octadecenol),linoleyl alcohol (cis-9,12-octadecadienol), γ-linolenyl alcohol(cis-6,9,12-octadecatrienol) and linolenyl alcohol(cis-9,12,15-octadecatrienol). In preferred embodiments, the fattyalcohol used in the compositions of the invention is cetyl, linolenylor, most preferably, oleyl alcohol.

In another embodiment, the pharmaceutical composition of the inventioncomprises an ester of a fatty alcohol as defined above with a C₁-C₆alkanoic acid such as acetic acid, propionic acid, butyric acid, valericacid and caproic acid.

In a further embodiment, the pharmaceutical composition of the inventioncomprises an ester of a saturated or cis-unsaturated C₁₀-C₂₀ fatty acidwith an alcohol selected from a C₂-C₈ alkanediol or glycerol, said esterbeing a monoester with said C₂-C₈ alkanediol or glycerol or a diesterwith glycerol.

The C₁₀-C₂₀ fatty acid is preferably a C₁₆-C₂₀, most preferably a C₁₈fatty acid. In one embodiment, the C₁₀-C₂₀ fatty acid is saturated suchas, but without being limited to, capric acid, lauric acid, myristicacid, palmitic acid, stearic acid and arachidic acid. In anotherembodiment, the C₁₀-C₂₀ fatty acid is a cis-unsaturated fatty acid suchas, but without being limited to, palmitoleic acid (cis-9-hexadecenoicacid), oleic acid (cis-9-octadecenoic acid), cis-vaccenic acid(cis-11-octadecenoic acid), linoleic acid (cis-9,12-octadecadienoicacid), γ-linolenic acid (cis-6,9,12-octadecatrienoic acid), linolenicacid (cis-9,12,15-octadecatrienoic acid) and arachidonic acid(cis-5,8,11,14-eicosatetraenoic acid).

According to the invention, the alkanediol has 2 to 8, preferably 2 to4, and more preferably, 2 carbon atoms, and is selected from, but notbeing limited to, 1,3-propanediol, 1,4-butanediol and, preferably,1,2-ethylene glycol. An example of such an ester is 1,2-ethylene glycolmonooleate.

According to another embodiment of the invention, the active ingredientof the pharmaceutical composition is a mono- or diester of glycerol withthe long-chain fatty acid. In one preferred embodiment, themonoglyceride is glycerol monooleate. The diglycerides contain one freehydroxyl group and the other two hydroxyl groups may be both esterifiedwith 2 molecules of the long-chain fatty acid, e.g. glycerol dioleate,or one of the hydroxyl groups is esterified with one molecule of thelong-chain fatty acid and a second hydroxyl group is esterified with aC₁-C₆ alkanoic acid such as acetic acid, propionic acid, butyric acid,valeric acid and caproic acid.

The immune system, in both its innate and adaptive arms, is involved inregulating inflammation of every type, and inflammation is a key factorin processes such as wound healing, connective tissue re-modeling,angiogenesis, organ regeneration, neuroprotection, as well as in theadaptive immune responses seen in autoimmunity, allergies, graftrejection, and infection (see Cohen, 2000; Schwartz and Cohen, 2000).Therefore, anti-inflammatory agents that modulate the inflammatoryresponse such as those described here will be useful in a variety ofconditions.

Inflammatory disorders that can be treated with the immunomodulators ofthe present invention include, but are not limited to,immunologically-mediated chronic or acute inflammatory disordersselected from an autoimmune disease, severe allergies, asthma, graftrejection or for the treatment of chronic degenerative diseases such asAlzheimer's disease, and in neuroprotection, organ regeneration, chroniculcers of the skin, and schizophrenia.

Examples of autoimmune diseases that can be treated according to theinvention are multiple sclerosis or a human arthritic condition, e.g.rheumatoid arthritis, reactive arthritis with Reiter's syndrome,ankylosing spondylitis and other inflammations of the joints mediated bythe immune system. Other autoimmune diseases are contemplated and arepresented in the following list in the context of the organ or tissueinvolved. Thus, according to the invention, the immunologically-mediatedinflammatory disorder may be myasthenia gravis, Guillain-Barré syndrome,and other inflammatory diseases of the nervous system; psoriasis,pemphigus vulgaris and other diseases of the skin; systemic lupuserythematosus, glomerulonephritis and other diseases affecting thekidneys; atherosclerosis and other inflammations of the blood vessels;autoimmune hepatitis, inflammatory bowel diseases, e.g. Crohn's disease,pancreatitis, and other conditions of the gastrointestinal system; type1 diabetes mellitus (insulin-dependent diabetes mellitus or IDDM),autoimmune thyroiditis (Hashimoto's thyroiditis), and other diseases ofthe endocrine system.

One of the models used to test the anti-inflammatory activity of theagents according to the invention is adjuvant arthritis (AA), anexperimental disease of the joints inducible in some strains of rats byimmunizing with Mycobacterium tuberculosis in complete Freund's adjuvant(CFA). These animals develop an arthritis whose features are similar tothose of rheumatoid arthritis in humans and thus serve as animal modelsof human arthritic conditions such as rheumatoid arthritis, reactivearthritis in Reiter's syndrome, ankylosing spondylitis and otherinflammations of the joints which appear to be mediated by the immunesystem (Pearson, 1964). Adjuvant arthritis also serves as a model ofimmune-mediated inflammation in general including cell-mediatedautoimmune reactions, graft rejection and allergic reaction. Forexample, treatments which can suppress rheumatoid arthritis includeimmunosuppressive agents such as corticosteroids, cyclosporin A (Jaffeeet al., 1989; Pollock et al., 1989), azathioprine, and otherimmunosuppressive agents which are broadly used in the treatment ofautoimmune diseases. Therefore, suppression of adjuvant arthritis by atherapeutic agent indicates that the agent is potentially useful as abroad anti-inflammatory agent.

The pharmaceutical composition provided by the present invention may bein solid, semisolid or liquid form and may further includepharmaceutically acceptable fillers, carriers or diluents, and otherinert ingredients and excipients. The composition can be administered byany suitable route such as, but not limited to, oral, topical, orparenteral e.g. by injection through subcutaneous, intravenous,intramuscular, or any other suitable route. Since many of the compoundsare oily, they are preferably administered parenterally, more preferablysubcutaneously. If given continuously, the compounds of the presentinvention are each typically administered by 1-4 injections per day orby continuous subcutaneous infusions, for example, using a mini-pump.The dosage will depend of the state of the patient and severity of thedisease and will be determined as deemed appropriate by thepractitioner.

For parenteral administration, the compounds may be formulated by mixingeach at the desired degree of purity, in a unit dosage injectable form(solution, suspension, or emulsion), with a pharmaceutically acceptablecarrier, i.e., one that is non-toxic to recipients at the dosages andconcentrations employed and is compatible with other ingredients of theformulation. Generally, the formulations are prepared by contacting thecompounds of the present invention each uniformly and intimately withliquid carriers or finely divided solid carriers or both. Then, ifnecessary, the product is shaped into the desired formulation.Preferably the carrier is a parenteral carrier, more preferably asolution that is isotonic with the blood of the recipient. Examples ofsuch carrier vehicles include water, saline, Ringer's solution, anddextrose solution. Non-aqueous vehicles such as fixed oils can be alsouseful, as well as liposomes. These preparations can be made byconventional methods known to those skilled in the art, for example asdescribed in “Remington's Pharmaceutical Science”, A. R. Gennaro, ed.,17th edition, 1985, Mack Publishing Company, Easton, Pa., USA.

The invention will now be illustrated by the following non-limitingexamples.

EXAMPLES Example 1 Anti-Inflammatory Effect of Oleyl Alcohol and OtherAgents—Protection Against Adjuvant Arthritis (AA)

AA was induced by immunizing inbred 8-10-week old Lewis rats(Harlan-Olac Limited, Blackthorn, Oxon, UK), at the base of the tailwith 1 mg/0.1 ml of killed Mycobacterium tuberculosis (Sigma) in IFA(Sigma) as described (Pearson, 1956). Arthritis of the limbs was notedto develop 12-14 days later and was scored on a scale of 0-16 summingthe severity of the inflammation of each of the 4 limbs on a scale of0-4, as described (Holoshitz et al., 1983). The peak of the arthritisusually was observed around day 26 after immunization.

Control rats were untreated or treated by injections of saline. Apositive control of immunosuppression was obtained by including a groupof rats treated with the corticosteroid agent dexamethasone (200 μg)administered intraperitoneally every other day beginning on day 12 afterinduction.

The immunomodulator of the invention (100 μl oleyl alcohol, glycerolmono-oleate, linolenyl alcohol or cetyl alcohol) was administeredsubcutaneously (SC) once 14 days before induction of AA or on day 12after induction of AA. The percent inhibition of inflammation measuredon the day of maximal inflammation was computed as follows:$\frac{{mean}\quad{maximal}\quad{score}\quad{of}\quad{test}\quad{group}}{{mean}\quad{maximal}\quad{score}\quad{of}\quad{control}\quad{group}} \times 100\quad\%$

All four compounds were found to be effective, producing more than 60%inhibition of inflammation whereas oleic acid had no effect. The resultsare summarized in Table 1.

Two further experiments showed that 500 μl of oleyl alcohol (100 μlcorresponds to about 90 mg oleyl alcohol) suppressed the inflammation by96% and 91%. TABLE 1 Effects of various agents on the inflammation ofadjuvant arthritis Compound Tested % Inhibition (100 μl) Glycerolmono-oleate 98% Oleyl alcohol 78% Linolenyl alcohol 75% Cetyl alcohol66%

Example 2 Protection Against AA by Different Doses of Oleyl Alcohol

To study the dose response effect of oleyl alcohol in AA, oleyl alcoholwas administered subcutaneously in doses of 10, 50, 100 or 500 mg toLewis rats once 14 days before induction of AA, as described in Example1 above.

FIG. 1 shows the dose response effect of oleyl alcohol. It can be seenthat increasing doses of oleyl alcohol suppressed the arthritis. On theday of peak disease, day 26, the inflammation was suppressed by 14% (10μl), 61% (50 μl), 78% (100 μl) and 90% (500 μl).

Example 3 Anti-Inflammatory Effect of Oleyl Alcohol and OtherImmunomodulators and Protection Against EAE in DA Rats

Experimental autoimmune encephalomyelitis (EAE) is an experimentalautoimmune disease inducible in some strains of rats by immunizationwith myelin basic protein (MBP) or proteolipid protein (PLP) in completeFreund's adjuvant (CFA) or with an emulsion of the rat's spinal cord ineither CFA or incomplete Freund's adjuvant (IFA). EAE in DA rats isconsidered as a model of chronic EAE. Within two to three weeks theanimals develop cellular infiltration of the myelin sheaths of thecentral nervous system resulting in demyelination and paralysis. Most ofthe animals die, but others have milder symptoms, and some animalsdevelop a chronic form of the disease that resembles chronic relapsingand remitting multiple sclerosis (MS) in humans. Therefore, theseanimals with EAE serve as a model for the human MS autoimmune disease.EAE develops in the animal about 12 days after immunization and ischaracterized by paralysis of various degrees due to inflammation of thecentral nervous system. In some strains, like the Lewis rat, theparalysis can last up to 6-7 days and the rats usually recover unlessthey die during the peak of their acute paralysis. In other strains ofrats like the DA rat, the paralysis can be chronic and remitting.

For the induction and clinical assessment of EAE, spinal cord obtainedfrom DA rats is frozen, thawed and minced thoroughly with a spatulabefore immunization. Rats are immunized by one subcutaneous injection(just under the skin) into the dorsal base of the tail with 200 μlemulsion prepared from 1:1 IFA (Difco, Detroit, Mich., USA) and antigen(volume/weight, i.e. 100 μl IFA/100 mg of whole spinal cord) or from 1:1CFA (IFA was complemented with 4 mg/ml of Mycobacterium tuberculosisstrain 37RA) and antigen (volume/weight, i.e. 100 μl CFA/100 mg of wholespinal cord). The emulsion was prepared by titration with a gas-tightglass syringe and a needle, 1.2 mm diameter. Rats are regularly weighedand examined for clinical signs of EAE. A four-graded scale was used toassess clinical severity: 0, no paralysis; 1, tail weakness (hanging);2, hind limb paralysis; 3, hind and fore limb paralysis; 4, severe totalparalysis (Lorentzen et al., 1995).

Groups of 5 or 7 DA strain female rats, 8-9 week old, are immunized inthe hind footpads with 0.1 ml per footpad of IFA containing 100 mg ofwhole, homogenized DA spinal cord, for a total of 200 mg per rat. On theday of immunization, the rats are treated by SC injection with oleylalcohol or other agent according to the invention (100 μl) or withparaffin oil (control). The rats are scored for EAE on a severity scaleof 0-4 as described above.

Example 4 Anti-Inflammatory Effect of Oleyl Alcohol and ProtectionAgainst EAE in Lewis Rats

EAE induced in Lewis rats is considered as a model of acute inflammationin the brain (as opposed to the chronic disease in DA rats).

For EAE induction, three lyophilized guinea pig spinal cord homogenate(GPSCH) emulsions were prepared as follows: (i) 25 mg of lyophilizedGPSCH (GP2) was suspended in 2.5 ml of sterile PBS (Sigma) and incubatedfor one hour at 37° C.; (ii) 54.1 mg of Mycobacterium tuberculosis H37Ra(MT, Difco) was suspended in 13.5 ml CFA (Sigma) containing 1 mg/ml MTto obtain 5 mg/ml MT; (iii) 2.5 ml CFA (5 mg/ml MT) was added into vialwith 2.5 ml of PBS containing 25 mg GPSCH to yield 5 mg/ml GPSCH and 2.5mg/ml MT. The mixture was transferred into a glass syringe connected toa second glass syringe through a Luer lock bridge. The material wasmixed well by transferring from one syringe to another for about 10minutes until the materiall was well emulsified. The emulsion of GPSCHat a dose of 1 mg/rat and MT at a dose of 0.5 mg/rat in CFA induced EAEin rats (based on previous titration).

For the treatment, two groups of eight 9-10 weeks old Lewis rats(Harlan, Israel), were treated with the test samples (oleyl alcohol orIFA) 14 days before EAE induction. The group treated with IFA served asthe control group. The test samples were injected at a dose of 0.5 ml/kgonce, subcutaneously. A third group of 8 rats was not treated and servedas non-treated control.

EAE was induced in rats of all three groups 14 days after injection ofthe test samples by injection with 0.1 ml of the GPSCH emulsion in CFAinto each of the hind leg foot pads (0.2 ml per rat).

The EAE clinical signs were observed and scored from the 9^(th) daypost-EAE induction until the termination of the experiment according tothe following five-graded scale to assess clinical severity: 0, normalbehavior; 1, weight loss; 2, tail weakness; 3, hind legs hypotonia andweakness; 4, hind legs paralysis; 4, severe total paralysis; 5, impairedrespiration and/or convulsions and/or full paralysis or death. All ratshaving scores of 1 and above were considered sick.

The calculation of EAE results was carried out as follows:

(i) Calculation of the Incidence of Disease

The number of sick animals in each group were summed. The incidence ofdisease and the % activity were calculated as follows:${{Incidence}\quad{of}\quad{disease}} = {\frac{{{No}.\quad{of}}\quad{sick}\quad{rats}\quad{in}\quad{group}}{{{No}.\quad{of}}\quad{rats}\quad{in}\quad{group}} \times 100\quad\%}$${\%\quad{activity}}{* =}{{1 - {\frac{\left( {{disease}\quad{incidence}\quad{in}\quad{treated}\quad{group}} \right)}{{disease}\quad{incidence}\quad{in}\quad{control}\quad{group}} \times 100\quad\%}}*=\left( {{according}\quad{to}\quad{incidence}} \right)}$(ii) Calculation of the Mean Maximal Score (MMS)

The maximal score of each rat in the group were summed. The mean maximalscore (MMS) and the % activity of the group were calculated as follows:${{Mean}\quad{Maximal}\quad{score}} = \frac{\sum{{Maximal}\quad{score}\quad{of}\quad{each}\quad{rat}}}{{{No}.\quad{of}}\quad{rats}\quad{in}\quad{the}\quad{group}}$${\%\quad{activity}}{* =}{{\left( {1 - \frac{{MMS}\quad{of}\quad{treated}\quad{group}}{{MMS}\quad{of}\quad{control}\quad{group}}} \right) \times 100}*=\left( {\%\quad{activity}\quad{according}\quad{to}\quad{MMS}} \right)}$(iii) Calculation of the Group Mean Score (GMS)

The mean score of each rat during the observation period were summed(score 5 was counted forward). The mean score of the group and its %activity were calculated as follows:${{Mean}\quad{score}}\quad = \frac{\sum{{Group}\quad{score}\quad{of}\quad{each}\quad{rat}}}{{{No}.\quad{of}}\quad{rats}\quad{in}\quad{the}\quad{group}}$${\%\quad{activity}}{* =}{{\left( {1 - \frac{{GMS}\quad{of}\quad{treated}\quad{group}}{{GMS}\quad{of}\quad{control}\quad{group}}} \right) \times 100}*=\left( {\%\quad{activity}\quad{according}\quad{to}\quad{GMS}} \right)}$(iv) Calculation of the Mean Onset of Disease

The time of disease onset (days) for each rat in the group were summed.The mean onset of disease for the group was calculated. The time ofonset of disease for those rats that did not develop EAE was consideredas 25 days (duration of study).

(v) Calculation of the Mean Duration of Disease

The disease duration (days) of each rat in each group were summed. Themean disease duration of the group was calculated. The disease durationof rats that did not develop EAE was considered as zero.

The evaluation of the clinical manifestations of EAE, i.e. % incidenceof disease, MMS, GMS, mean duration and onset of EAE disease issummarized in Table 2. The graphs of the disease profile for each groupare presented in FIGS. 2 and 3 for treatment with oleyl alcohol and IFA,respectively.

As shown by the results, no essential difference in incidence of disease(62.5% to 75% incidence) or mean maximum score (1.75 to 2.38 MMS) wasobserved between the IFA-injected groups and non-treated control group.Oleyl alcohol showed a beneficial effect on all the clinical parametersthat were tested. It exhibited 77.1% activity according to group meanscore (GMS) and 63% activity according to mean maximum score (MMS)compared to the non-treated control group. The mean onset of disease was18.6 days in the oleyl alcohol treated group compared to 15.5 days inthe non-treated control group. The duration of disease was 2.0 days inthe oleyl alcohol treated group compared to 5.13 days in the non-treatedcontrol group. The duration of the EAE clinical signs in the testedgroups was between 1 and 7 days, except one rat in the group treatedwith IFA. IFA had minor effect, if any, on the rat EAE. No mortality wasobserved in the tested groups, except one rat in the non-treated controlgroup. TABLE 2 Evaluation EAE clinical results Mean % % % Onset DiseaseGroup Test % Activity Activity Activity of disease duration No. SampleIncidence Incidence MMS MMS GMS GMS (Day No.) (days) 1 OA 50.0% 33.3%0.88 63.0% 0.22 77.1% 18.6 2.0 2 IFA 62.5% 16.7% 1.75 26.5% 0.52 45.8%17.0 3.75 3 NTC 75.0% NA 2.38 NA 0.96 NA 15.5 5.13OA—Oleyl alcohol;NTC—non-treated control;NA—Not applicable

Example 5 Effect of Oleyl Alcohol on Skin Allograft Survival

The immune system represents a strong barrier for successfultransplantation of organs or tissues between non-genetically identicaldonor and recipient. Both CD4⁺ and CD8⁺ T cells participate in graftrejection.

Skin graft transplantation is carried out essentially as describedbefore (Birk et al., 1999). Thus, mice are shaved and 1 cm² sections ofskin are cut from the dorsal side of sacrificed donors and cleaned inPBS. Two patches of dorsal skin, 1 cm² each, are cut from theanesthetized recipients (Nembutal 6 mg/ml, 0.25 ml/mouse) in preparationfor the allograft. Two donor allografts per recipient are grafted ontothe dorsal lesioned patches. Histoacryl (B. Braun Melsungen AG,Melsungen, Germany) is applied around the graft. Nobecutan (ASTR, AstraTech, Glos G15, UK) is sprayed over the grafts.

In the experiment, groups of 6 BALB/c female mice, 8-week old, aregrafted with 1 cm², full thickness skin grafts from C57BL/6 female mice,8-week old. On the day of grafting, a group of recipient mice is treatedeither with paraffin oil or SC with 100 μl oleyl alcohol or anotherimmunomodulator according to the invention. The day of rejection isscored. The transplanted skin in the mice treated with theimmunomodulator survives longer in comparison with the untreated controlmice.

Example 6 Prevention and Treatment of SLE

Systemic lupus erythematosus is an autoimmune disease in which bothautoantibodies and immune complexes are involved. In order to test theimmunomodulators of the invention, mice with experimental SLE or(NZBxNZW)F1 mice that spontaneously develop autoimmune diseases thatclosely resemble SLE, can be used.

In order to induce experimental SLE, BALB/c mice are immunized with thehuman or murine anti-DNA monoclonal antibody 16/6Id (20 μg/mouse) in CFAin the hind footpads and boosted 3 weeks later with the same amount ofthe immunizing antibody in PBS. The mice are then tested forautoantibody production and clinical manifestations characteristic ofexperimental SLE. In order to either prevent induction of experimentalSLE or to cure mice afflicted with the disease, mice are given oleylalcohol or another immunomodulator according to the inventionsubcutaneously (100 μl per mouse) before or concomitant with theimmunization and some weeks after immunization. The number of injectionsis based on the effect of the tested compound on the disease inductionand progression. The animals are regularly weighed and examined forclinical signs of SLE as described, for example, in WO 96/30057.

Example 7 Prevention and Treatment of Autoimmune Thyroiditis

Experimental autoimmune thyroiditis (EAT) can be induced in a number ofanimals by immunizing with thyroglobulin in CFA. Both humoral antibodiesand T_(DTH) cells directed against the thyrogllobulin develop, resultingin thyroid inflammation. EAT appears to best mimic Hashimoto'sthyroiditis.

EAT is induced as previously described (Rose et al., 1971) by injectingeach mouse subcutaneously with thyroglobulin extract obtained from onethyroid gland. The extract is emulsified in IFA (Difco Laboratories,Detroit, Mich.), to which are added 7 mg/ml Mycobacterium tuberculosis,H37Ra strain (Difco Laboratories). This injection is repeated one weeklater. Donors of thyroglobulin extract are mice of the C3H/eB strain.4-5 weeks later, EAT is assayed by removing thyroid glands of recipientmice, fixing them in 10% formalin solution and then in 70% alcohol, andexamining microscopic sections stained with hematoxylin and eosin.Microscopic slides are coded and examined without knowledge of theiridentity. A diagnosis of EAT is made by observing at least oneunequivocal focus of infiltration by mononuclear cells. Treatment isperformed by injecting SC oleyl alcohol or another immunomodulator (100μl per animal) before induction of EAT, concomitant with or thereafter(control animals are injected paraffin oil), and the animals areregularly weighed and examined for clinical signs of EAT by knownconventional methods.

REFERENCES

-   Birk et al., 1999. The 60 kDa heat shock protein modulates allograft    rejection. Proc. Nat. Acad. Science USA. 96: 5159-63.-   Calder, P C. 1998. Immunoregulatory and anti-inflammatory effects of    n-3 polyunsaturated fatty acids. Braz. J. Med. Biol. Res. 31:467-90.-   Cohen, I R. 2000, Tending Adam's Garden: Evolving the Cognitive    Immune Self. Academic Press, London, UK.-   Cohen, I R. 2000. “Discrimination and Dialogue in the Immune    System”. Seminars in Immunology. 12: 215-219; 269-271; 321-323.-   Holoshitz, Y. et al., 1983. “Lines of T lymphocytes mediate or    vaccinate against autoimmune arthritis”, Science 219: 56.-   Jaffee, B. D. et al., 1989, “The effect of immunomodulating drugs on    adjuvant-induced arthritis in Lewis rats”, Agents Actions. 27 (3-4):    344-6.-   Lorentzen J. C. et al., 1995, “Protracted, relapsing and    demyelinating experimental autoimmune encephalomyelitis in DA rats    immunized with syngeneic spinal cord and incomplete Freund's    adjuvant”, J. Neuroimmunology. 63: 193-205.-   Pearson, C M. 1964, “Experimental models in rheumatoid disease”,    Arthritis Rheum. 7:80.-   Pearson, C M. 1956, “Development of arthritis, periarthritis and    periostitis in rats given adjuvant”, Proc. Soc. Exp. Biol. Med.    91:91.-   Pollock et al., 1989, “Pharmacokinetic analysis of cyclosporine in    adjuvant arthritic rats”, Drug Metab. Dispos. 17 (6): 595-9.-   Rodriguez, M. D., Gamez, R., Sanchez, M and H. Garcia. 1998, J.    Appl. Toxicol. 18: 313-6.-   Rose, N. R., F. J. Twarog and A. J. Crowe. 1971. Murine thryoiditis:    importance of adjuvant and mouse strain for the induction of thyroid    lesions. J. Immunol. 106:698)-   Schwartz, M. and Cohen. I R. 2000, “Autoimmunity can benefit    self-maintenance”. Immunol Today. 21:265-8.-   The Merck Index, 13th Edition, 2001, Merck & Co. Inc., Rahway, N.J.,    U.S.A.

1-40. (canceled)
 41. A method for the treatment of inflammation, particularly immunologically-mediated inflammation, which comprises administering to a patient in need an effective amount of an immunomodulator selected from: (a) a saturated or cis-unsaturated C₁₀-C₂₀ fatty alcohol or an ester thereof with a C₁-C₆ alkanoic acid; (b) a monoester of a C₂-C₈ alkanediol or of glycerol with a saturated or cis-unsaturated C₁₀-C₂₀ fatty acid; and (c) a diester of glycerol with a saturated or cis-unsaturated C₁₀-C₂₀ fatty acid.
 42. A method according to claim 41, wherein said immunomodulator is a saturated C₁₀-C₁₂ fatty alcohol.
 43. The method according to claim 42, wherein said saturated C₁₀-C₂₀ fatty alcohol is selected from decyl alcohol, lauryl alcohol, myristyl alcohol, cetyl alcohol and stearyl alcohol.
 44. The method according to claim 41, wherein said immunomodulator is a cis-unsaturated C₁₆-C₁₈ fatty alcohol.
 45. The method according to claim 44, wherein the cis-unsaturated C₁₆-C₁₈ fatty alcohol is selected from oleyl alcohol, linoleyl alcohol, y-linolenyl alcohol and linolenyl alcohol.
 46. The method according to claim 41, wherein the immunomodulator is an ester of a saturated or cis-unsaturated C₁₀-C₂₀ fatty alcohol with a C₂-C₆ alkanoic acid.
 47. The method according to claim 41, wherein said immunomodulator is a monoester of a saturated or cis-unsaturated C₁₀-C₂₀ fatty acid with a C₂-C₈ alkanediol.
 48. The method according to claim 47, wherein said alkanediol is selected from 1,2-ethylene glycol, 1,3-propanediol and 1,4-butanediol.
 49. The method according to claim 41, wherein said immunomodulator is a monoester of glycerol with a saturated or cis-unsaturated C₁₀-C₂₀ fatty acid.
 50. The method according to claim 41, wherein said immunomodulator is a diester of glycerol with a saturated or cis-unsaturated C₁₀-C₂₀ fatty acid.
 51. The method according to claim 41, wherein said fatty acid is a saturated C₁₀-C₂₀ fatty acid.
 52. The method according to claim 51, wherein said saturated C₁₀-C₂₀ fatty acid is selected from capric acid, lauric acid, myristic acid, palmitic acid, stearic acid and arachidic acid.
 53. The method according to claim 41, wherein said fatty acid is a cis-unsaturated C₁₀-C₂₀ fatty acid.
 54. The method according to claim 53, wherein said cis-unsaturated Ci_(o)-C₂o fatty acid is selected from palmitoleic acid, oleic acid, cis-vaccenic acid, linoleic acid, y-linolenic acid, linolenic acid, and arachidonic acid.
 55. The method according to claim 54, wherein said immunomodulator is glyceryl monooleate.
 56. The method according to claim 54, wherein said immunomodulator is glyceryl dioleate.
 57. A method according to claim 41 for the treatment of immunologically-mediated inflammatory disorders selected from an autoimmune disease, severe allergies, asthma, graft rejection or for the treatment of chronic degenerative diseases such as Alzheimer's disease, and in neuroprotection, organ regeneration, chronic ulcers of the skin, and schizophrenia.
 58. The method according to claim 57, wherein said autoimmune disease is multiple sclerosis or a human arthritic condition.
 59. The method according to claim 58, wherein said human arthritic condition is selected from rheumatoid arthritis, reactive arthritis with Reiter's syndrome, ankylosing spondylitis and other inflammations of the joints mediated by the immune system.
 60. The method according to claim 57, wherein said immunologically-mediated inflammatory disorder is myasthenia gravis, Guillain Barre syndrome, and other inflammatory diseases of the nervous system; psoriasis, pemphigus vulgaris and other diseases of the skin; systemic lupus erythematosus, glomerulonephritis and other diseases affecting the kidneys; atherosclerosis and other inflammations of the blood vessels, autoimmune hepatitis, inflammatory bowel diseases, pancreatitis, and other conditions of the gastrointestinal system; type 1 diabetes mellitus, thyroiditis, and other diseases of the endocrine system. 